Compositions and methods for regulating RNA translation via CD154 CA-dinucleotide repeat

ABSTRACT

Compositions and methods for regulating CD154 gene expression are provided that rely on the interaction of hnRNP L with the CA-dinucleotide rich sequence of the 3′-untranslated region of CD154.

This application is a continuation-in-part application ofPCT/US2008/075965 filed Sep. 11, 2008, which is a continuation-in-partapplication of PCT/US2006/035260, filed Sep. 11, 2006, which claimsbenefit of U.S. Provisional Patent Application Ser. No. 60/716,708,filed Sep. 13, 2005. This application is also a continuation-in-part ofSer. No. 12/064,471 filed Feb. 22, 2008 and a continuation-in-part ofSer. No. 11/854,148 filed Sep. 12, 2007, which both also claim benefitof U.S. Provisional Patent Application Ser. No. 60/716,708, filed Sep.13, 2005, the contents of which are incorporated herein by reference intheir entireties.

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant No. AI34928). The U.S.government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The expression of CD154 (CD40 ligand), a member of the Tumor NecrosisFactor (TNF) gene family, by activated T lymphocytes is critical in thedevelopment of humoral and cell-mediated immunity (Foy, et al. (1996)Annu. Rev. Immunol. 14:591-617; Grewal & Flavell (1998) Ann. Rev.Immunol. 16:111-135; Hollenbaugh, et al. (1994) Immunol. Rev. 138:23-37;Noelle (1996) Immunity 4:415-419). CD154 blockade retards thedevelopment and progression of immune responses in an array oftransplantation and autoimmune disease models ranging from SystemicLupus Erythematosus to Rheumatoid Arthritis to Multiple Sclerosis (Foy,et al. (1996) supra; Grewal & Flavell (1998) supra). Resting T cellsexpress little or no CD154 (Lane, et al. (1992) Eur. J. Immunol.22:2573-2578; Nusslein, et al. (1996) Eur. J. Immunol. 26:846-850; Roy,et al. (1993) J. Immunol. 151:2497-2510) and signals (anti-CD3,mitogenic lectins) that trigger resting T cells to engage in high levelsof proliferation and cytokine production, elicit little (CD4+ T cells)or no (CD8+ T cells) expression on either mouse or human T cells (Lane,et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al. (1993)supra), suggesting different pathways of gene regulation. Maximalexpression of CD154 requires pharmacologic stimulation provided byphorbol myristate acetate (PMA) and calcium ionophores such as ionomycin(Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al.(1993) supra; Roy, et al. (1994) Eur. J. Immunol. 25:596-603).Cyclosporine and glucocorticoids block CD154 induction on T lymphocytes;these effects are presumed to be transcriptional (Fuleihan, et al.(1994) J. Clin. Invest. 93:1315-1320; Roy, et al. (1993) supra), basedon the presence of NF-AT sites in the CD154 promoter (Schubert, et al.(1995) J. Biol. Chem. 15:29264-29627). Since cyclosporine andglucocorticoids also inhibit cytokine production (Ashwell, et al. (1992)Ann. Rev. Immunol. 18:309-345; Sigal & Dumont (1992) Ann. Rev. Immunol.10:519-60), this pathway does not account for the differentialregulation of CD154 expression by T lymphocytes.

CD154 mRNA has been shown to be unstable in activated T lymphocytes,with a half-life (−30 minutes) approximating that seen withinterleukins-2 (IL-2; Ford, et al. (1999) J. Immunol. 162:4037-4044;Murakami, et al. (1999) J. Immunol. 163:2667-2673; Rigby, et al. (1999)J. Immunol. 163:4199-4206; Suarez, et al. (1997) Eur. J. Immunol.27:2822-2829). Nevertheless, several studies indicate that cytokine(IL-2 and TNF-alpha) and CD154 mRNA stability and expression areregulated through distinct pathways (Ford, et al. (1999) supra;Lindsten, et al. (1989) Science 244:339-343; Murakami, et al. (1999)supra). A region (nucleotides 468-835 referenced to the translationalstop site) within the 986 nucleotide human CD154 3′-untranslated region(3′-UTR) confers an increase in the rate of mRNA turnover to chimericreporter gene constructs in vivo (Hamilton, et al. (2003) Mol. Cell.Biol.; 23(2):510-25). This region lacks canonical AURE-type sequences,containing a polypyrimidine rich element as well as CA-dinucleotiderepeat and polycytidine sequences. Members of the human polypyrimidinetract binding protein (PTB) gene family were identified and shown todirectly interact with cytidines and uridines within this region(Hamilton, et al. (2003) supra; kosinski, et al. (2003) J. Immunol.170(2):979-88), consistent with the presence of multiple PTB consensusbinding sites (Anwar, et al. (2000) J. Biol. Chem. 275:34231-34235;Singh, et al. (1995) Science 268:1173-1176). Overexpression of spliceisoforms of the PTB proteins differentially regulates CD154 expressionand mRNA accumulation in a 3′-UTR-dependent manner in cell lines andnormal human T cells. These effects are specific and restricted toreporter constructs containing the 3′-UTR polypyrimidine rich region.

The murine CD154 (mCD154) 3′-UTR is ˜0.3 kb shorter than its humancounterpart, due to a 292 nucleotide insertion present at the 5′ end ofthe human 3′-UTR. The remaining portion of the human and entire mCD1543′-UTR exhibits 70% conservation with retention of the polycytidine,polypyrimidine, and CA-dinucleotide repeat regions as well as an AUREthat is found immediately 5′ of the polyadenylation signal sequence.Murine CD154 3′-UTR inhibits luciferase mRNA accumulation and proteinactivity in a comparable manner relative to that seen with the humanCD154 3′-UTR. Further, deletion of the polypyrimidine-rich regioncis-acting element enhances inhibition of 3′-UTR-dependent geneexpression.

A novel pathway has now been identified which regulates translation ofCD154 mRNA.

SUMMARY OF THE INVENTION

The present invention embraces methods for modulating the translation ofa ribonucleic acid that contains CA-dinucleotide rich sequences.Identification of this sequence and its function due to hnRNP Linteractions raises the possibility that small molecule inhibitors canbe identified that can regulate its activity in the context of the CD1543′-UTR or any other gene that contains a similar sequence. The methodsinvolve contacting a cell or tissue containing a CA-dinucleotide richsequence of the CD154 mRNA 3′-untranslated region operatively-linked toa ribonucleic acid with an agent that binds to the CA-dinucleotide richsequence of the CD154 mRNA 3′-untranslated region or an agent whichmodulates the level or activity of an hnRNP L protein so that thenuclear export or translation of the ribonucleic acid is modulated.

The present invention also encompasses methods for preventing ortreating a disease or condition associated with CD154-CD40 interactions.These methods involve administering to a subject in need of treatment anagent which binds to a CA-dinucleotide rich sequence of the CD154 mRNA3′-untranslated region or an agent which modulates the level or activityof hnRNP L protein so that CD154 translation is inhibited therebypreventing or treating the disease or condition associated withCD154-CD40 interactions.

The present invention further provides a method for identifying agentsthat modulate the level or activity of hnRNP L. This method of theinvention involves contacting a test cell containing hnRNP L protein,and a CA-dinucleotide rich sequence of the CD154 mRNA 3′-untranslatedregion operatively-linked to a nucleic acid encoding a reporter protein,with an agent and detecting reporter protein expression in the testcell. A decrease in reporter protein expression in the test cellcontacted with the agent relative to reporter protein expression in atest cell not contacted with the agent, indicates that the agentincreases the level or activity of hnRNP L in the cell. An increase inreporter protein expression in the test cell contacted with the agentrelative to reporter protein expression in a test cell not contactedwith the agent, indicates that the agent decreases the level or activityof hnRNP L in the cell.

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The human and murine CD154 3′-UTR are highly conserved, except for thepresence of a 293 nucleotide insertion immediately after thetranslational stop site. Most notable are the presence of adjacent CU-and CA-rich regions. In addition, the murine CD154 3′-UTR containspolycytidine and AU-rich element sequences that are expanded relative toits human counterpart. The CA-rich region is of interest as itrepresents an extended series of CA-dinucleotide repeats, which arealmost always intronic. Chimeric reporter gene constructs have indicatedthat a cis-acting element of the human CD154 3′-UTR maps to a regioncontaining both the CU- and CA-rich domains. This effect was present inmultiple cell lines (Jurkat, HeLa) as well as normal human T cells.

To delineate the mCD154 3′-UTR sequences involved in regulating CD154expression, chimeric luciferase reporter gene constructs were generatedand transiently transfected into HeLa cells. The presence of the mCD1543′-UTR markedly reduced luciferase expression. Prior studies with thehuman CD154 3′-UTR indicate that cytoplasmic levels of PTB proteinsregulate the function of this element. The binding specificity of PTBproteins, among other factors, indicate that the CU-rich domain (alsoreferred to herein as CURE) is the major cis-acting element in the humanCD154 3′-UTR. However, deletion of the CURE from mCD154 3′-UTR resultedin a decrease rather than an increase in luciferase activity. Deletionof the CA-rich domain (also referred to herein as CARE) provided asimilar effect. Accordingly, both the CURE and CARE domains in the CD1543′-UTR function as cis-acting elements.

To eliminate the possibility that deletion of either the CURE or CAREdomains enhances the function of a secondary cis-acting element, boththe CURE and CARE domains were deleted. This mutation increasedluciferase activity to 171% of that seen with control. Superimposingmutation of the polycytidine (poly C) or ARE sequences in the context ofdeleting the CURE and CARE elements in the CD154 3′-UTR had noadditional effect on luciferase expression. These data indicate that theCURE and CARE regions each function as cis-acting elements to regulateexpression of CD154. An identical pattern was observed with transienttransfection of human peripheral blood mononuclear cells (PBMC) underbasal and activated conditions. Insertion of the CURE (CARE+) or CARE(CARE+) alone in the 3′UTR of reporter genes established that eachcis-acting element was sufficient to reduce luciferase activity andpoly(A)+ mRNA levels. Thus, the effects of both the CURE and the CAREalone are transferable in the 3′UTR of heterologous transcripts.

Following transient transfection of HeLa cells, RNA was extracted andluciferase mRNA levels quantified by real-time RT-PCR. Deletion ofeither the CURE or the CARE region had no significant effect onsteady-state mRNA levels. Deletion of both CURE and CARE regionsincreased luciferase mRNA expression. These data indicated that both theCURE and CARE regions regulated CD154 mRNA turnover. Using a HeLaTet-Off™ system, identical constructs were generated containing eitherthe CURE or CARE region. HeLa cells were transiently transfectedovernight and transcription was inhibited by the addition ofdoxycycline. Cytoplasmic RNA was collected at time 0 and at varioustimes thereafter. Luciferase mRNA levels measured by real-time RT-PCRindicated that at time 0, steady state levels of cytoplasmic luciferasemRNA were reduced by the presence of CD154 3′-UTR containing either theCURE or CARE regions. The presence of the mCD154 3′-UTR increasedluciferase mRNA turnover relative to controls, as seen with the human3′-UTR. Deletion of both CURE and CARE regions increased luciferase mRNAstability to that of controls. Similarly, the CARE-deletion (CARE−)still exhibited an increased rate of decay, indicating a role of theretained CURE in mRNA decay. Surprisingly, deletion of the CURE (CURE−)element increased the stability of cytoplasmic poly (A)+ mRNA to that ofthe control vector, although it inhibited luciferase expression andpoly(A)+ mRNA accumulation. These data indicated that the retained CAREfunctioned independently of mRNA decay to limit steady state levels ofluciferase poly(A) RNA.

To determine how the CARE region reduces cytoplasmic mRNA levels in theabsence of effects on mRNA stability, the contribution of other portionsof the mCD154 3′-UTR were eliminated. Isolated CURE and/or CARE regionswere cloned into the 3′-UTR of the pTRE vector and luciferase activityand mRNA turnover examined. This analysis indicated that the CURE alone(CURE+) conferred increased mRNA decay, establishing that it alonepromotes cytoplasmic mRNA instability. When combined with the observedstability of the CURE− reporter, these data indicate that theinstability associated with the CD154 3′-UTR derives from the CURE. Incontrast, the CARE element alone (CARE+) had no effect on the decay ofpoly(A)+ mRNA, despite reducing luciferase RNA to an equivalent degree.Thus, both in isolation as well as in the context of the CD154 3′-UTR(CURE−), the CARE reduces luciferase activity and poly(A)+ mRNA levelsindependent of cytoplasmic decay.

The 3′-UTR CARE reduced poly (A)+ mRNA levels without increasing mRNAdecay, prompting examination of its function as a transcriptionalsilencer when placed downstream of the reporter gene. No effect wasseen. Since prior measurements of reporter mRNA by quantitative RT-PCRrelied on the use of cytoplasmic poly(A)+ mRNA, it was determinedwhether these results were potentially influenced by an effect of the3′-UTR CARE on mRNA polyadenylation. Total cytoplasmic RNA was analyzedby RT-PCR in which the effect of priming with either oligo d(T) orrandom hexamers was compared. The 3′-UTR CURE alone reduced luciferasemRNA levels independent of priming, consistent with its effects oncytoplasmic mRNA decay. In contrast, the 3′-UTR CARE alone increasedby >3-fold the apparent levels of input luciferase RNA seen with randomhexamer priming relative to that seen with oligo d(T). Similar resultswere seen in which luciferase-specific priming of reverse transcriptionwas used instead of random hexamers.

Since oligo d(T)-, but not random hexamer-dependent priming was affectedby the 3′-UTR CARE, it was determined whether luciferase mRNApolyadenyation was affected using Ligase-Mediated Polyadenylation TailAssay (LM-PAT) of cytoplasmic RNA (Salles, et al. (1999) Methods.17:38-45). The presence of the 3′-UTR CARE reduced the poly(A) tail to<50 adenylates, relative to the control and 3′-UTR CURE reporter mRNAwhere poly (A) tails as long as 150 bases were seen. The effect of the3′-UTR CARE on poly(A) tail length was equally apparent in both nuclearand cytoplasmic extracts. These data indicate that the effect of the3′-UTR CARE on poly(A) tail length is transduced in the nucleus.Finally, it was demonstrated that CD154 mRNA polyadenylation isregulated in normal murine T cells. Under resting and early (6 hours)activation conditions, nearly all CD154 mRNA was found to bedeadenylated. At 24 hours, CD154 mRNA poly(A) tail length increased.Thus, CD154 mRNA polyadenylation is regulated as a function ofactivation. This was not a generalized effect on all mRNA, asexamination of TNF mRNA exhibited a distinct pattern of modulation ofpoly(A) tail length.

Experiments were performed to characterize, purify and identify theprotein(s) binding to and regulating expression of CD154 via the CAREsequence. Using activated human peripheral blood mononuclear cells,immunoprecipitation followed by RNA extraction and RT-PCR establishedthat PTB and hnRNP L each bind native CD154 mRNA in vivo. GAPDH RNA wasnot found in these immunoprecipitates, establishing the specificity ofthis interaction. Moreover, immunoblotting demonstrated that PTB andhnRNP L coprecipitate. The specificity of the hnRNP L interaction withthe murine CD154 3′-UTR and CARE in vivo was examined. Followingtransient transfection of HeLa cells with reporter vectors that lacked(control) or contained these sequences, hnRNP L immunoprecipitates wereanalyzed for the presence of luciferase mRNA. Luciferase mRNA wascoprecipitated with hnRNP L in a 3′-UTR CARE-dependent manner. Thespecificity of this interaction of hnRNP L was shown both by the absenceor GAPDH or a luciferase mRNA lacking a 3′-UTR CARE in theimmunoprecipitates.

The functional significance of the hnRNP L-3′-UTR CARE interaction wastested. Knockdowns of hnRNP L levels by RNA interference eliminated theinhibitory effects of the 3′-UTR CARE on luciferase expression and poly(A) tail length. Overexpression of hnRNP L strikingly enhanced theactivity of the 3′-UTR CARE, increasing the inhibition of luciferaseactivity from ˜50% to 90%. This enhanced suppression was accompanied byincreased luciferase mRNA deadenylation as measured by oligo d(T)priming or LM-PAT assays. These results indicated that hnRNP L binds the3′-UTR CARE in vivo to regulate nuclear polyadenylation. Thus, whencytoplasmic levels of hnRNP L are low, CD154 mRNA is translated moreefficiently and increased surface expression of CD154 results.

These data demonstrate the existence of a novel pathway of mRNA turnoverregulation. Additionally, these data indicate that the polymorphicnature of CARE in CD154 3′-UTR may influence CD154 expression and immuneresponses. The presence of CARE could influence mRNA biogenesis both atthe level of splicing and mRNA stability. Further, the relative levelsof cytoplasmic hnRNP L appear to be regulated by specific stimuli andmodulating the levels of hnRNP L could be used as a means of modulatingthe expression of CD154 at the level of translation and nuclear export.Accordingly, inhibiting CD154 expression at the level of hnRNP Lexpression or activity or the CARE is useful in autoimmune andinflammatory diseases, whereas enhancing expression of CD154 bytargeting this pathway could be used in immunotherapy of cancer or foraugmenting immune responses in immunodeficient individuals. Inparticular, it is contemplated that several potential pathways may leadto increased CD154 mRNA polyadenylation as a function of duration of Tcell activation. These include a decline in nuclear levels of hnRNP L orits ability to interact with the CARE. Alternatively, the ability ofhnRNP L molecules to homodimerize may be influenced by T cellactivation, perhaps due to post-translational modification. Third,prolonged T cell activation is associated with increases in the levelsof PTB proteins and increased levels of PTB might limit hnRNP L-hnRNP Lhomodimer formation. Each mechanism may limit recruitment ofdeadenylases or polyadenylation factors to the mRNA transcript.

Precise regulation of CD154 is critical in immunoregulation; transgenicoverexpression of CD154 results in a phenotype suggestive of SystemicLupus Erythematosus (SLE) (Clegg, et al. (1997) Int. Immunol.9(8):1111-22; Dunn, et al. (1997) J. Histochem. Cytochem. 45(1):129-41;Mehling, et al. (2001) J. Exp. Med. 194(5):615-28; Higuchi, et al.(2002) J. Immunol. 168(1):9-12). T cells from patients with SLE exhibitenhanced surface expression of CD154, particularly after 24 hours(Koshy, et al. (1996) J. Clin. Invest. 98(3):826-37). The 3′-UTR CARE ispolymorphic in humans and increased CA repeats (>24) have been reportedto occur at increased frequency in patients with SLE (Citores, et al.(2004) Ann. Rheum. Dis. 63(3):310-7). Characterization of the functionof this cis-acting element and its regulation by hnRNP L provides anovel means to treat this disease.

Moreover, soluble CD154 derived from platelets has been associated withboth acute coronary syndromes as well as increased risk forcardiovascular disease. Thus, targeting hnRNP L or the CARE may beuseful in treatment of acute and chronic atherosclerotic diseaseincluding angina, myocardial infarction, stroke and other conditions ofacute or chronic vascular insufficiency.

Thus, the present invention embraces methods of modulating nuclearexport or translation of a ribonucleic acid molecule operatively-linkedto a CARE sequence of the CD154 mRNA 3′-untranslated region using anagent which binds to the CARE sequence or which modulates the level oractivity of an hnRNP L protein so that the nuclear export or translationof the ribonucleic acid is modulated. Operatively-linked is intended tomean that the ribonucleic acid is linked to the CARE sequence in amanner which allows for translation of the ribonucleic acid molecule tobe regulated by the CARE sequence, i.e., the ribonucleic acid moleculeand CARE sequence are located on the same transcript.

As used herein, the CARE sequence of the CD154 mRNA 3′-untranslatedregion (3′-UTR) is located between nucleotides 468 to 835 of the humanCD154 3′-UTR cDNA relative to the translational stop site; i.e., withinthe BstNI-HphI restriction enzyme fragment of the 3′-UTR. This region isset forth herein as SEQ ID NO:1:

Caggctctagaacgtctaacacagtggagaaccgaaacccccccccccccccccgccaccctctcggacagttattcattctctttcaatctctctctctccatctctctctttcagtctctctctctcaacctctttcttccaatctctctttctcaatctctctgtttccctttgtcagtctcttccctcccccagtctctcttctctccccctttctaacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacagagtcaggccgttgctagtcagttctcttctttccaccctgtccctatctctaccactatagatgagggtgaggagtag (SEQ ID NO:1), wherein the CARE repeat is underlinedand located at positions 223 to 286 of SEQ ID NO:1.

Methods of modulating or regulating translation of an RNA moleculeoperatively-linked to a CARE sequence of a CD154 3′-UTR encompass bothenhancing and inhibiting the translation of said RNA molecule. Bindingof an agent, e.g., an siRNA or hnRNP L (GENBANK Accession No.NP_(—)001005335 or NP_(—)001524) to the CARE or increasing theexpression or activity of hnRNP L via an agent results in inhibition oftranslation of the RNA molecule. Conversely, decreasing the expressionor activity of hnRNP L via an agent (e.g., an siRNA) enhancestranslation of the RNA molecule. Effects on translation of the RNA canbe determined using standard techniques such as western blot analysis ofthe translated product of the RNA sequence, or if the protein beingtranslated is an enzyme, enzymatic assays can be performed. Inparticular embodiments, the ribonucleic acid molecule encodes CD154. Assuch, binding of an agent to the CARE or increasing the level oractivity of hnRNP L by pharmacological agents is contemplated as auseful tool in the treatment of autoimmune and inflammatory diseaseswhich are associated with CD154-CD40 interactions, whereas decreasingthe level or activity of hnRNP L by pharmacological agents iscontemplated as a useful tool in the treatment of, e.g., cancer, whereinCD40 activation by CD154 is advantageous.

Thus, the present invention also encompasses methods for preventing ortreating a disease or condition associated with B cell CD154-CD40interactions, i.e., diseases or conditions resulting from enhanced CD40activation by CD154, or diseases or conditions associated with lack ofCD40 activation by CD154. The methods involve administering to a subjectin need of treatment an agent which binds to a CA-dinucleotide richsequence of the CD154 mRNA 3′-untranslated region or an agent thatincreases the level or activity of hnRNP L protein so that CD154translation is inhibited thereby preventing or treating the disease orcondition associated with CD40 activation by CD154. Diseases orconditions which can be prevented or treated in accordance with theinstant method, include, but are not limited to, allograft rejection;allergy (including anaphylaxis); atherosclerosis including angina,myocardial infarction, stroke and other conditions of chronic or acutevascular insufficiency; autoimmune conditions including drug-inducedlupus, systemic lupus erythematosus, adult rheumatoid arthritis,juvenile rheumatoid arthritis, scleroderma, Sjogren's Syndrome, etc.;and viral diseases that involve B-cells, including Epstein-Barrinfection, cancer, and retroviral infection including infection with ahuman immunodeficiency virus. Because it has been suggested that B cellactivation is associated with the induction of human immunodeficiencyvirus replication from latency, it may be desirable to decreasetranslation of CD154 mRNA in HIV positive individuals who have not yetdeveloped AIDS or ARC.

In particular embodiments, the subject is a primate, such as a human. Inother embodiments, the subject is a mammal of commercial importance, ora companion animal or other animal of value. Thus, subjects alsoinclude, but are not limited to, sheep, horses, cattle, goats, pigs,dogs, cats, rabbits, guinea pigs, hamsters, rats and mice.

It is contemplated that the agent can be administered as a capsule,intramuscularly, intraperitoneally, subcutaneously, intradermally orapplied locally to a wound site. It is also clear that the invention canbe used with a skin graft procedure. The skin is a notoriously difficulttissue with which to achieve or maintain engraftment. A preferred routeof administration for treating or preventing skin graft rejection istopical, subdermal, intradermal or subcutaneous, though systemic andother routes are also contemplated.

Another route of administration for skin graft includes directapplication locally (by topical application, immersion or bath, or localinjection) into the subject tissue bed, or to the graft tissue itself.High local concentrations of the agent, particularly in areas oflymphatic drainage, are expected to be particularly advantageous.Alternatively, the graft tissue can be transfected or transformed with arecombinant expression vector to overexpress hnRNP L.

An effective amount of an agent which binds to a CARE sequence of theCD154 mRNA 3′-untranslated region or an agent that alters the level oractivity of hnRNP L protein is an amount which decreases or inhibits thesigns or symptoms of diseases or conditions associated with CD40activation (e.g., edema, fever, and loss of graft function) and will bedependent on the nature of the agent.

Agents useful in accordance with the methods provided herein include,but are not limited to, purified hnRNP L protein, a recombinantexpression vector expressing hnRNP L, a recombinant expression vectorexpressing an siRNA which binds the CARE sequence or hnRNP L RNA,organic molecules, biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

An isolated or purified protein hnRNP L protein for administrating PTBor PTB-T protein administration to a cell or tissue can be produced byvarious means. An isolated or purified protein is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the hnRNP L protein is derived. To besubstantially free of cellular material includes preparations of hnRNP Lprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. When thehnRNP L protein is recombinantly produced, it is also preferablysubstantially free of culture medium.

Recombinant production of hnRNP L protein typically involves generatinga fusion protein such as a GST-hnRNP L in which the hnRNP L proteinsequence is fused to the C-terminus of the GST sequence. Such fusionproteins can facilitate the purification of recombinant hnRNP L protein.Alternatively, the fusion protein is a hnRNP L protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of hnRNP Lprotein can be increased through use of a heterologous signal sequence.Preferably, a hnRNP L chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation.Alternatively, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers or PCR amplification.PCR amplification of gene fragments can be carried out using anchorprimers which give rise to complementary overhangs between twoconsecutive gene fragments which are subsequently annealed andreamplified to generate a chimeric gene sequence (see, e.g., CurrentProtocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A hnRNPL-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the hnRNP L protein.

A recombinant expression vector contains a nucleic acid encoding hnRNP Lin a form suitable for expression of the nucleic acid in a host cell,which means that the recombinant expression vector includes one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operatively-linked to the nucleic acid to beexpressed. Within a recombinant expression vector, operatively-linked isintended to mean that the nucleotide sequence of interest is linked tothe regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell). A regulatory sequence is intended to includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are described, forexample, in Goeddel (1990) Methods Enzymol. 185:3-7. Regulatorysequences include those which direct constitutive expression of anucleic acid sequence in many types of host cells and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by one ofskill in the art that the design of the expression vector depends onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorcan be introduced into a host cell to thereby produce proteins orpeptides of hnRNP L, isoforms of hnRNP L, mutant forms of hnRNP L,fusion proteins, and the like.

A recombinant expression vector can be designed for expression of hnRNPL protein in prokaryotic or eukaryotic cells. For example, hnRNP Lproteins can be expressed in bacterial cells such as E. coli, insectcells (using baculovirus expression vectors), yeast cells or mammaliancells. Suitable host cells are discussed further in Goeddel (1990)supra. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve to increase expression of recombinant protein;increase the solubility of the recombinant protein; and aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann, et al., (1988) Gene 69:301-315) and pET ld(Studier, et al. (1990) Methods Enzymol. 185:60-89). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene underthe transcriptional control of the lacUV 5 promoter.

A yeast expression vector is also contemplated. Examples of vectors forexpression in yeast Saccharomyces cerevisiae include pYepSec 1 (Baldari,et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982)Cell 30:933-943), pJRY88 (Schultz, et al. (1987) Gene 54:113-123), pYES2(Invitrogen™ Corp., San Diego, Calif.), and picZ (Invitrogen™ Corp., SanDiego, Calif.).

Alternatively, hnRNP L protein can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith, et al. (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

Further, nucleic acid molecules encoding hnRNP L are expressed inmammalian cells using a mammalian expression vector. As will beappreciated by one of skill in the art, hnRNP L expression in mammaliancells provides a means of purifying the proteins as well as a means ofmodulating the endogenous levels of hnRNP L protein in a cell. Examplesof mammalian expression vectors include any one of the well-knownrecombinant viral vectors, pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman, et al. (1987) EMBO J. 6:187-195). When used in mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.For other suitable expression systems for both prokaryotic andeukaryotic cells see chapters 16 and 17 of Sambrook, et al. MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The recombinant mammalian expression vector may further be capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart. Non-limiting examples of suitable tissue-specific promoters includethe albumin promoter (liver-specific; Pinkert, et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji, et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund, et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and EP 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

In addition to increasing the expression of hnRNP L to modulate thelevels of hnRNP L present in the cell, hnRNP L expression can bedecreased to modulate the levels of hnRNP L present in the cell. Thus, arecombinant expression vector harboring a nucleic acid encoding hnRNP L,or an iRNA target sequence thereof, cloned into the expression vector inan antisense orientation is also provided. That is, the nucleic acidencoding hnRNP L, or a target fragment thereof, is operatively-linked toa regulatory sequence in a manner which allows for expression (bytranscription of the nucleic acid sequence) of an RNA molecule which isantisense to hnRNP L mRNA. Regulatory sequences operatively-linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen which direct constitutive,tissue-specific or cell type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, et al. (1986)Reviews-Trends in Genetics Vol. 1(1).

Host cells into which a hnRNP L nucleic acid can be introduced, e.g., ahnRNP L nucleic acid within a vector (e.g., a recombinant expressionvector) or a hnRNP L nucleic acid containing sequences which allow it tohomologously recombined into a specific site of the host cell's genome,are further contemplated. The terms host cell and recombinant host cellare used interchangeably herein. It is understood that such terms refernot only to the particular subject cell but to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms transformation and transfection are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. supra and otherlaboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acids encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an hnRNP L protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die). A host cell, such as a prokaryotic or eukaryotic host cellin culture, can be used to produce (i.e., express) an hnRNP L protein.

The host cells can also be used to produce non-human transgenic animals.For example, a host cell is a fertilized oocyte or an embryonic stemcell into which hnRNP L-coding sequences have been introduced. Such hostcells can then be used to create non-human transgenic animals in whichexogenous hnRNP L sequences have been introduced into their genome orhomologous recombinant animals in which endogenous hnRNP L sequenceshave been altered. Such animals are useful for studying the functionand/or activity of an hnRNP L protein and for identifying and/orevaluating modulators of hnRNP L activity. As used herein, a transgenicanimal is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a homologousrecombinant animal is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous hnRNP L gene has been alteredby homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal can be created by introducing a hnRNP L-encodingnucleic acid into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection or retroviral infection, and allowing the oocyte todevelop in a pseudopregnant female foster animal. Alternatively, anon-human homologue of a human hnRNP L gene, such as a rat or mousehnRNP L gene, can be used as a transgene. Intronic sequences andpolyadenylation signals may also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operatively-linked to ahnRNP L transgene to direct expression of a hnRNP L protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866; 4,870,009; 4,873,191; and in Hogan, Manipulatingthe Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1986). Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of an hnRNP L transgene in its genome and/orexpression of hnRNP L mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a hnRNP L protein can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an hnRNP L gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the hnRNP L gene. The hnRNP L gene can be a humangene or a non-human homologue of a human hnRNP L gene. For example, amouse hnRNP L gene can be used to construct a homologous recombinationnucleic acid molecule, e.g., a vector, suitable for altering anendogenous hnRNP L gene in the mouse genome. The homologousrecombination nucleic acid molecule may be designed such that, uponhomologous recombination, the endogenous hnRNP L gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a knock out vector). Alternatively, the homologous recombinationnucleic acid molecule can be designed such that, upon homologousrecombination, the endogenous hnRNP L gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous hnRNP L protein). In the homologous recombination nucleicacid molecule, the altered portion of the hnRNP L gene is flanked at its5′ and 3′ ends by additional nucleic acid sequence of the hnRNP L geneto allow for homologous recombination to occur between the exogenoushnRNP L gene carried by the homologous recombination nucleic acidmolecule and an endogenous hnRNP L gene in a cell, e.g., an embryonicstem cell or fetal fibroblast. The additional flanking hnRNP L nucleicacid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas and Capecchi(1987) Cell 51:503). The homologous recombination nucleic acid moleculeis introduced into a cell, e.g., an embryonic stem cell line, by forexample electroporation, and cells in which the introduced hnRNP L genehas homologously recombined with the endogenous hnRNP L gene areselected (see, e.g., Li, et al. (1992) Cell 69:915). The selected cellscan then be injected into a blastocyst of an animal (e.g., a mouse) toform aggregation chimeras (see, e.g., Bradley, In: Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, E. J. ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals arewell-known (see, e.g., Bradley (1991) Current Opin. Biotechnol.2:823-829; WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

A method for identifying an agent that modulates the level or activityof hnRNP L is also encompassed by the instant invention. The methodinvolves contacting a test cell containing hnRNP L protein, and aCA-dinucleotide rich sequence of the CD154 mRNA 3′-untranslated regionoperatively-linked to a nucleic acid encoding a reporter protein, withan agent and detecting reporter protein expression in the test cell. Adecrease in reporter protein expression in the test cell contacted withthe agent relative to reporter protein expression in a test cell notcontacted with the agent, indicates that the agent increases the levelor activity of hnRNP L in the cell. Conversely, an increase in reporterprotein expression in the test cell contacted with the agent relative toreporter protein expression in a test cell not contacted with the agent,indicates that the agent decreases the level or activity of hnRNP L inthe cell. Test cells expressing a reporter which can be used inaccordance with the method of the invention are, in certain embodiments,mammalian cells including human cells.

The reporter gene sequence(s) can be inserted into a recombinantexpression vector according to methods disclosed herein. More than onereporter gene can be inserted into the construct such that the testcells containing the resulting construct can be assayed by differentmeans. The test cells which contain the nucleic acid encoding thereporter and which express the reporter can be identified by at leastfour general approaches; detecting DNA-DNA or DNA-RNA hybridization;observing the presence or absence of marker gene functions (e.g.,resistance to antibiotics); assessing the level of transcription asmeasured by the expression of reporter mRNA transcripts in the hostcell; and detecting the reporter gene product as measured by immunoassayor by its biological activity.

The test cells can be cultured under standard conditions of temperature,incubation time, optical density, plating density and media compositioncorresponding to the nutritional and physiological requirements of thecells. However, conditions for maintenance and growth of the test cellcan be different from those for assaying candidate test compounds in thescreening methods of the invention. Modified culture conditions andmedia are used to facilitate detection of the expression of a reportermolecule. Any techniques known in the art can be applied to establishthe optimal conditions.

A reporter gene refers to any genetic sequence that is detectable anddistinguishable from other genetic sequences present in test cells.Desirably, the reporter nucleic acid encodes a protein that is readilydetectable either by its presence, or by its activity that results inthe generation of a detectable signal. A nucleic acid encoding thereporter is used in the invention to monitor and report the translationof an RNA operatively-linked to a CA-dinucleotide rich sequence of aCD154 3′-untranslated region in test cells.

A variety of enzymes can be used as reporters including, but are notlimited to, β-galactosidase (Nolan, et al. (1988) Proc. Natl. Acad. Sci.USA 85:2603-2607), chloramphenicol acetyltransferase (CAT; Gorman, etal. (1982) Molecular Cell Biology 2:1044; Prost, et al. (1986) Gene45:107-111), β-lactamase, β-glucuronidase and alkaline phosphatase(Berger, et al. (1988) Gene 66:1-10; Cullen, et al. (1992) MethodsEnzymol. 216:362-368). Transcription of the reporter gene leads toproduction of the enzyme in test cells. The amount of enzyme present canbe measured via its enzymatic action on a substrate resulting in theformation of a detectable reaction product. The methods of the inventionprovide means for determining the amount of reaction product, whereinthe amount of reaction product generated or the remaining amount ofsubstrate is related to the amount of enzyme activity. For some enzymes,such as β-galactosidase, β-glucuronidase and β-lactamase, well-knownfluorogenic substrates are available that allow the enzyme to covertsuch substrates into detectable fluorescent products.

A variety of bioluminescent, chemiluminescent and fluorescent proteinscan also be used as light-emitting reporters in the invention. Exemplarylight-emitting reporters, which are enzymes and require cofactor(s) toemit light, include, but are not limited to, the bacterial luciferase(luxAB gene product) of Vibrio harveyi (Karp (1989) Biochim. Biophys.Acta 1007:84-90; Stewart, et al. (1992) J. Gen. Microbiol.138:1289-1300), and the luciferase from firefly, Photinus pyralis (DeWet, et al. (1987) Mol. Cell. Biol. 7:725-737).

Other types of light-emitting reporter, which do not require substratesor cofactors, are wild-type green fluorescent protein (GFP) of Victoriaaequoria (Chalfie, et al. (1994) Science 263:802-805), modified GFPs(Heim, et al. (1995) Nature 373:663-4; WO 96/23810), and the geneproducts encoded by the Photorhabdus luminescens lux operon (luxABCDE)(Francis, et al. (2000) Infect. Immun. 68(6):3594-600). Transcriptionand translation of these types of reporter genes leads to theaccumulation of the fluorescent or bioluminescent proteins in testcells, which can be measured by a device, such as a fluorimeter, flowcytometer, or luminometer. Methods for performing assays on fluorescentmaterials are well-known in the art (e.g., Lackowicz, 1983, Principlesof Fluorescence Spectroscopy, New York, Plenum Press).

For convenience and efficiency, enzymatic reporters and light-emittingreporters are desirable for the screening assays of the invention.Accordingly, the invention encompasses histochemical, colorimetric andfluorometric assays. An exemplary reporter construct, exemplifiedherein, contains the CA-dinucleotide rich sequence of a CD1543′-untranslated region which regulates the translation of and thereforethe expression of the reporter luciferase.

By way of illustration, a screening assay of the invention can becarried out by culturing a test cell containing a nucleic acid encodingluciferase operatively-linked to a CARE sequence of a CD1543′-untranslated region; adding a test agent to a point of application,such as a well, in the plate and incubating the plate for a timesufficient to allow the test agent to effect luciferase mRNAtranslation; detecting luminescence of the test cells contacted with thetest agent, wherein luminescence indicates expression of the luciferasepolypeptide in the test cells; and comparing the luminescence of testcells not contacted with the test agent. A decrease in luminescence ofthe test cell contacting the test agent relative to the luminescence oftest cells not contacting the test agent indicates that the test agentcauses a decrease in the level or activity of hnRNP L. An increase inluminescence of the test cell contacting the test agent relative to theluminescence of test cells not contacting the test agent indicates thatthe test agent causes an increase in the level or activity of hnRNP L.

Agents which can be screened using the method provided herein encompassnumerous chemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 daltons. Agents encompass functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The agents often contain cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Agents canalso be found among biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Agents are obtained from awide variety of sources including libraries of natural or syntheticcompounds.

A variety of other reagents such as salts and neutral proteins can beincluded in the screening assays. Also, reagents that otherwise improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, and the like can be used. The mixtureof components can be added in any order that provides for the requisitebinding.

Alternatively, antibodies against the hnRNP L can serve as the agent toinhibit (antagonize) or stimulate (agonize) hnRNP L activity. WholehnRNP L or an epitope bearing fragment thereof can be used as animmunogen to produce antibodies immunospecific for hnRNP L. Varioustechniques well-known in the art can be used routinely to produceantibodies (Kohler and Milstein (1975) Nature 256:495-497; Kozbor, etal. (1983) Immunol. Today 4:72; Cole, et al. (1985) In: MonoclonalAntibodies and Cancer Therapy, pp 77-96).

Example 1 cDNA and siRNA Plasmids

The pcDNA 3.1/LUC and tetracycline responsive vector pTRE-LUC utilizethe bovine growth hormone and beta globin polyadenylation signalsequences, respectively, and are known in the art (Hamilton, et al.(2003) Mol. Cell. Biol. 23:510-525). The murine CD154 3′-UTR correspondsto nucleotides (nt) 23-650 relative to the translational stop site wasamplified from total cellular RNA from B6 splenocytes activated with PMA(10 ng/ml; Sigma, St. Louis, Mo.)+Ionomycin (IONO, 0.5 μM) and clonedinto TOPO 2.1 (Invitrogen, Carlsbad, Calif.). Sequencing confirmedidentity with GenBank Gene ID No. 560692. Deletion of the CURE (nt127-228) or the CARE (nt 229-306) are referred to as CURE- and CARE−,respectively, while the CURE and CARE (nt 127-306) deletion is referredto as CU/CARE−. All were generated by QuikChange (Stratagene, La Jolla,Calif.) deletion from TOPO 2.1 as were the polycytidine and AREmutations in the context of the CU/CARE- and confirmed by sequencing.These sequences were released by EcoRI and cloned into the XbaI site inpcDNA 3.1 firefly luciferase (pcDNA3.1/LUC) vector (Hamilton, et al.(2003) supra). For reporters containing only CURE (CURE+) and CARE(CARE+), the CURE or CARE was deleted from the CU/CARE− in TOPO 2.1using QuikChange. The CURE and CARE were released from TOPO 2.1 andcloned into pcDNA 3.1 luciferase as described above. Generation ofpTRE-Luc vectors containing the murine CD154 3′-UTR were released byEcoRI digestion from TOPO vectors described above and cloned into theEcoRV site downstream of the luciferase coding region. The CARE wascloned downstream of the polyadenylation signal sequence in the BamHIsite of pGL3-promoter vector (Promega, Madison, Wis.).

SiRNA cDNA constructs targeting hnRNP L were purchased from Origene(Rockville, Md.); their activity was confirmed in HeLa cells byimmunoblot analysis. Generation of pcDNA3.1-hnRNP L was achieved by therelease of hnRNP L from pFASTBac hnRNP L through EcoRI and XhoIdigestion and cloning into EcoRI and XhoI site in pcDNA 3.1

Example 2 Transient Transfection Assay of Reporter Gene Activity

Except for siRNA and hnRNP L overexpression studies, HeLa cells weretransfected with 50 ng luciferase vectors, 1.5 μl lipofectamine(Invitrogen) and 4 μl PLUS (Invitrogen) in 0.5 ml RPMI for 3.5 hours at37° C. 5% CO2, after which 0.5 mL RPMI+20% FCS was added. After 20hours, cells were lysed and luciferase activity determined byluminometry. Individual experiments were analyzed for 3′-UTR-specificeffects by dividing the mean luciferase activity from triplicatetransfections of pcDNA3.1/LUC- or pTRE-LUC-based expression plasmids bythat obtained from cells transfected with the corresponding controlvector, which was assigned a value of 100%. In siRNA and hnRNP Loverexpression experiments, cells were transiently transfected at day −2with either 500 ng HuSH 303 or HuSH L79 or 250 ng empty pcDNA 3.1control vector or pcDNA 3.1 hnRNP L followed by a repeat cotransfectionof these plasmids along with the corresponding luciferase reporters atday 0, Separate cultures received equivalent amounts of thecorresponding control (empty vector or containing an irrelevant RNAsequence) vector on day −2 and 0. Transient transfection of human PBMCwas performed using AMAXA Nucleofection. After being transientlytransfected overnight, luciferase activity was measured. Additionalcultures were activated with PMA/IONO for 4 hours prior to analysis ofluciferase activity.

Example 3 RNA Analysis by Quantitative RT-PCR

Cytoplasmic RNA was extracted according to known methods (Gough (1988)Anal. Biochem. 173:93-9). Nuclei were pelleted and resuspended inSolution 1 (10 mM Tris, 150 mM NaCl, 1.5 mM MgCl2 and 0.65% NP-40) andspun through a 30% sucrose cushion. Nuclear RNA was then extracted(Chomczynski & Sacchi (1987) Anal. Biochem. 162:156-159). Poly (A) RNAwas purified using Oligotex beads (Qiagen, Valencia, Calif.). SubsequentqPCR analysis of input luciferase mRNA levels were measured usingestablished methods (Hamilton, et al. (2003) supra). For studies of mRNAstability, Tet-Off HeLa cells (Clontech, Mountain View, Calif.) werepurchased and carried according to manufacturer's instructions andtransiently transfected as described above, allowed to recoverovernight, then treated with Doxycycline (1 μg/ml) to shut offtranscription for specified times. Analysis of the effects of priming ongene expression utilized cytoplasmic RNA that was digested withTurboDNase I (Ambion, Austin, Tex.) then reverse transcribed with eitheroligo d(T), random hexamers, or a luciferase-specific primer (5′-TTT GGCGGT TGT TAC TTG AC-3′; SEQ ID NO:2) and Superscript II RT (Invitrogen).Reverse transcription reactions were analyzed for luciferase transcriptsusing 5′-GGT GGC TCC CGC TGA ATT GG-3′ (SEQ ID NO:3) and 5′-CCG TCA TCGTCT TTC CGT GC-3′ (SEQ ID NO:4) primers. Oligo dT reverse transcriptionswere analyzed for GAPDH transcripts RNA to control for input RNA(forward primer, 5′-acc acc ttc ttg atg tca tc-3′ (SEQ ID NO:5) andreverse primer, 5′-CAA GGC TGT GGG CAA GGT CA-3′ (SEQ ID NO:6)). RandomHexamer reverse transcriptions were analyzed for H4 histone RNA tocontrol for input RNA (forward primer, 5′-CAA Cat tca ggg cat cac caa-3′(SEQ ID NO:7) and reverse primer 5′-CCC GAA TCA CAT TCT CCA AGA A-3′(SEQ ID NO:8)) and IQ SYBR Green Supermix (Bio-rad) by real-time PCRusing a Bio-Rad iCycler. The luciferase/GAPDH or H4 transcript ratio wascalculated for each sample, where Ct=threshold cycle and DCt=LuciferaseCt−GAPDH (dT primed reverse transcription) or H4 (Random Hexamer primedreverse transcription) Ct. DDCt=DCt1-DCt2, where DCt1 is CD154 and DCt2is control. Fold Difference=2-DDCt.

In these experiments, the percent inhibition of CD154 3′-UTR-dependentluciferase expression seen with each vector and priming method wascalculated and then divided by the inhibition seen with the emptycontrol vector, which was assigned a value of 100%. In some instances,data was presented where the DCt obtained with oligo (dT) priming for agiven transfection was subtracted from that obtained with random hexamerpriming (DCtRH.-DCtdT).

Example 4 Immunoprecipitation Analysis

HeLa cells were transiently transfected as specified and culturedovernight or human PBMC were activated for 24 hours with PMA 10 ng/mland IONO 1 μM. Cytoplasmic and nuclear extracts were obtained usingconventional methods (Rigby, et al. (1999) J. Immunol. 163:4199-206),with the addition of Protector RNase (Roche, Indianapolis, Ind.).Extracts were immunoprecipitated in parallel with 4D11 (anti-RNP L) andBB7 (anti-PTB) antibodies as well as a mouse IgG isotype control boundto protein-A Sepharose beads (Pharmacia, Piscataway, N.J.). Beads werewashed six times in 150 mM NaCl, boiled in SDS-PAGE loading buffer andresolved by 12% SDS-PAGE and immunoblotted. Remaining beads weredigested with proteinase K (Roche), then extracted withphenol-chloroform. Following DNase I digestion, the presence of humanCD154 or luciferase mRNA in each precipitation was measured by oligod(T)-based reverse transcription and qPCR. Human CD154 primers included5′-TTG CGG GCA ACA ATC CAT TCA CTT-3′ (SEQ ID NO:9) and 5′-GTG GGC TTAACC GCT GTG CTG TAT T-3′ (SEQ ID NO:10).

Example 5 Analysis of Poly(A) Tail Length

For native CD154 mRNA polyadenylation assay, murine T cells werepurified from a B6 spleen or volunteer donor by negative selection usingthe EasySep® Mouse T Cell Enrichment Kit (StemCell Technologies), andsubsequently activated with CD3/CD28 (Dynal). Poly (A) tail length wasmeasured by LM-PAT assay (Salles, et al. (1999) supra), in which the 3′end of the poly (A) tail was hybridized to a primer containing oligo(dT)16 5′-GCG AGC TCC GCG GCC GCG (T)16-3′ (SEQ ID NO:11) containing aGC ‘anchor’ sequence (Operon). Target mRNA (100 ng) was incubated andwith phosphorylated oligo(dT)16 (Roche) at an unfavorable annealingtemperature (42° C.) in the presence of T4 DNA ligase (Invitrogen)saturating the poly (A) tail, thereby creating an oligo (dT) copy of thepoly A tail. At 42° C., the 3′ end of the poly (A) tail remains largelyunpaired due to unfavorable hybridization conditions. The oligo (dT)-GCanchor sequence was added at 10-fold molar excess and the temperaturereduced to 12° C., enabling selective hybridization to the unpaired 3′ends. Reverse transcription was performed (Superscript II ReverseTranscriptase, Invitrogen) followed by PCR using a primer correspondingto the GC-rich sequence in the oligo-(dT) anchor along with a primerspecific for the mRNA to be analyzed. Primers used for LM-PAT assayincluded: Luciferase, 5′-GCC ATC TGT TGT TTG CC-3′ (SEQ ID NO:12);mCD154, 5′-CTG TCT ACA GCA CTG TCG GG-3′ (SEQ ID NO:13); mTNF, 5′-CACCTG GCC TCT CTA CCT TG-3′ (SEQ ID NO:14). Primers were designed so thatthe PCR product encoded a restriction enzyme sites MnlI, AseI and HphI,respectively. Products were resolved by agarose gel electrophoresis andthe identity of the visualized band confirmed by restriction enzymedigestion.

1. A method for inhibiting the nuclear export or translation of aribonucleic acid comprising: contacting an isolated cell or tissuecontaining a CA-dinucleotide rich sequence of the CD154 mRNA3′-untranslated region operatively-linked to a heterologous ribonucleicacid with an agent which binds to the CA-dinucleotide rich sequence,wherein said agent is selected from the group consisting of a purifiedhnRNP L protein, and a recombinant expression vector expressing hnRNP Lprotein, and a recombinant expression vector expressing an siRNA whichbinds the CA-dinucleotide rich sequence or hnRNP L, so that the nuclearexport or translation of the ribonucleic acid is inhibited.